Copper/zinc-Superoxide dismutase is required for oxytetracycline resistance of Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae, along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Delta mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 microg ml(-1), whereas concentrations of up to 1,000 microg ml(-1) had no effect on the growth of the wild type. OTC resistance was restored in the sod1Delta mutant by complementation with wild-type SOD1. The effect of OTC appeared to be cytotoxic and was not evident in a ctt1Delta (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1Delta strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1Delta mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.

FIG. 1

Sensitivity of S. cerevisiae DJY122 (sod1Δ) to OTC. (A) Exponential-phase S. cerevisiae S150-2B (wild type) (○) and DJY122 (sod1Δ) (●) cultures were plated on YEPD agar supplemented with OTC. (B) OTC resistance was restored to DJY122 by complementation with plasmids pVC734 (centromeric; □) and YEp600 (multicopy; ■) bearing wild-type SOD1 sequences. CFUs determined after 5 days are expressed as percentages of values obtained in the absence of OTC. Shown are means from two sets of triplicate determinations from independent experiments ± standard errors of the means (n = 6) where these values exceed the dimensions of the symbols.

FIG. 2

OTC uptake by S. cerevisiae. Exponential-phase cultures of S. cerevisiae S150-2B (○) and DJY122 (●) in YEPD medium were supplemented with OTC at 100 μg ml⁻¹. Uptake was calculated from residual OTC concentrations in the medium and by reference to control incubation mixtures lacking cells. Shown are means from sextuplet determinations ± standard errors of the means. Typical results from one of three independent experiments are shown.

FIG. 3

Effect of OTC on the growth of S. cerevisiae DJY122 in YEPD broth. Cells were incubated in YEPD in either the absence (○, □) or presence (●, ■) of OTC (100 μg ml⁻¹) (circles and squares distinguish staggered cultures). Colony-forming ability was determined at intervals by plating samples on YEPD agar lacking OTC. Typical results from one of six independent experiments are shown. Points represent means from triplicate determinations. Standard errors of the means were smaller than the dimensions of the symbols.

FIG. 4

OTC-induced lipid peroxidation. Exponential-phase S. cerevisiae S150-2B (A) and DJY122 (B) cultures were incubated in MES buffer (pH 5.5)–1% (vol/vol) glucose in either the absence (open symbols) or presence (solid symbols) of OTC (100 μg ml⁻¹). Shown are mean values for lipid peroxidation from two sets of triplicate determinations from independent experiments ± standard errors of the means (n = 6).

FIG. 5

OTC-induced protein oxidation. Exponential-phase cultures of S. cerevisiae S150-2B and DJY122 in YEPD medium were supplemented with OTC at 100 μg ml⁻¹. (A) Total protein carbonyls as reflected by anti-DNP reactivities in cellular protein extracts. (B) Quantitative analysis of results shown in panel A. □, S150-2B; ■, DJY122. Typical results from one of three independent experiments are shown.